Maintenance of genetic integrity is critical for both cell viability and the suppression of human disease, including cancer. Therefore, the DNA damage response (DDR) has evolved to combat the myriad threats that the genome encounters on a daily basis e.g. genotoxins arising in the environment or as byproducts of cellular metabolism. Given the fundamental importance of the DDR in disease suppression, it has been the subject of intense investigation, resulting in the identification of a well-defined set of core DDR protein. However, the equally important questions of how these core DDR factors are recruited to, and cleared from, DNA lesions to promote DNA repair remains largely unanswered. Studies so far have revealed a complex cascade of posttranslational modifications (PTMs) e.g. phosphorylation, ubiquitination and sumoylation that drives DDR protein dynamics; but in most cases, key mechanistic detail is missing. We address this critical deficiency in our proposal, by building on our recent discoveries of (i) a SUMO-targeted ubiquitin ligase (STUbL) that selectively ubiquitinates SUMO chain modified proteins to generate a dual PTM signal and (ii) the unanticipated recognition of this SUMO-ubiquitin signal by the molecular segregase Cdc48(p97)-Ufd1-Npl4 (Cdc48-UN). This functional coupling of STUbL activity to the Cdc48-UN segregase motor suggests a novel mechanism for the dynamic control of DDR proteins by SUMO and ubiquitin i.e. through the selective extraction of SUMO-ubiquitin co-modified proteins from DNA lesions, coupled or not to proteasomal degradation. Consistent with this, inactivating STUbL or Cdc48-UN, both of which localize to DNA lesions, disrupts normal DDR protein dynamics and causes severe genome instability. Herein, we propose to define the specific joint targets and mechanisms of STUbL and Cdc48-UN in the DDR through our three synergistic Specific Aims. In Aim 1, we will delineate co-functions for STUbL and Cdc48-UN in remodeling the proteome at topoisomerase 1 (Top1)-DNA adducts, which is crucial for the repair of this highly genotoxic lesion. In Aim 2, we will determine the cooperative mechanisms of STUbL and Cdc48-UN that promote the DDR elicited by de-protected telomeres in fission yeast. In humans, such telomere de-protection occurs spontaneously e.g. during ageing, and can be tumorigenic. In Aim 3, exemplifying the strength of pioneering analyses in fission yeast (Aim 2), we reveal that STUbL also promotes the DDR at de-protected mammalian telomeres. We will define the key STUbL targets in this DDR and determine if they are also p97 (Cdc48) targets. These focused Aims will be achieved collaboratively using innovative genetic, cell biological, biochemical and proteomic approaches in both fission yeast and mammalian cells. Results will provide mechanistic insight into how SUMO and ubiquitin signaling is integrated at DNA lesions to remodel the proteome, and thereby promote the DDR. Moreover, as STUbL mediates the therapeutic effect of arsenic trioxide in leukemia, and p97 (Cdc48) is also being targeted in disease therapy, our results will critically guide the development and improvement of such therapies.